JP5903968B2 - Method and apparatus for evaluating cell culture substrate, and method for producing cell culture substrate - Google Patents

Description

  The present invention relates to a method and an apparatus for evaluating a cell culture substrate having a functional layer on the substrate, and a method for producing such a cell culture substrate.

  A technique called “cell sheet engineering”, in which cells are cultured in a sheet form and the cells are recovered in a sheet form simply by lowering the temperature without using an enzyme such as trypsin, has attracted attention in the field of regenerative medicine. The cell sheet obtained by this technology has already been confirmed to have a certain therapeutic effect in regenerative medicine such as the cornea and periodontal tissue, and clinical research and clinical trials are already underway in Europe. It is also possible to create a three-dimensional tissue model by laminating multiple types of cell sheets, and to create mature tissue with vascular tissue in vitro. Research and development and treatment based on this technology Is expected more and more in the future.

  Control of adhesion and desorption of the cultured cell sheet is performed, for example, via a functional layer such as poly-N-isopropylacrylamide (PIPAAm) fixed on a substrate. This polymer is a material whose hydration ability changes mainly in response to temperature. At a temperature lower than the critical dissolution temperature, the affinity for surrounding water is improved, and the polymer takes up water and swells, so that cells are swelled on the surface. It exhibits the property of making it difficult to adhere (cell non-adhesiveness), and the property that makes it easier for cells to adhere to the surface due to water detaching from the polymer at temperatures above the same temperature (cell adhesion) It is. It is known that cell deadhesion is affected by the immobilization density and chain length of the above PIPAAm. Therefore, in order to examine the quality of cell deadhesion, a method for evaluating the state of the functional layer Development is desired.

As a conventional method for evaluating a cell culture substrate provided with a functional layer on the substrate, (Patent Document 1) is known. In (Patent Document 1), cells are seeded on the surface of a cell culture substrate coated with a temperature-responsive polymer such as poly-N-isopropylacrylamide whose hydration power changes in a temperature range of 0 to 80 ° C. In 48 hours, temperature-responsive cell culture in which cells are detached and recovered from the substrate surface by temperature treatment without using protein hydrolase and quantified with a spectrometer using a reagent that reacts with living cells A method for evaluating a substrate is disclosed. The cultured cells are required to be attached on the surface of the base material in an individual state so as to be 100 to 10,000 cells / cm ² per surface area of the base material. That is, when cells are collected in a sheet state, the influence of the force of pulling the cells due to cell-cell adhesion cannot be ignored. The result of eliminating the influence of cell-cell adhesion and reflecting only the interaction between the substrate and the cells is obtained.

  The evaluation method described in the above (Patent Document 1) has a problem in that it takes at least several hours to several tens of hours to perform the evaluation in order to perform cell culture.

JP 2009-82123 A

  Therefore, in view of the above-described conventional situation, the present invention eliminates the need for cell culture and can evaluate the state of the functional layer composed of PIPAAm and the like in a short time, and evaluation for implementing the method. An object is to provide an apparatus. Another object of the present invention is to provide a method for producing a cell culture substrate having a good functional layer state using the above evaluation method.

When the present inventor further injects liquid into the droplets supplied to the surface of the functional layer to expand the volume of the droplets or sucks the liquid to contract the volume of the droplets, the PIPAAm or the like is used. The inventors focused on the fact that the expansion or contraction behavior differs depending on the state of the functional layer for culturing cells. Then, it was found that the state of the functional layer can be evaluated by observing a phenomenon (stick-slip phenomenon) in which the contact angle of the droplet repeatedly increases and decreases during the process of expanding or contracting the droplet and analyzing the observation result. The present invention has been completed. That is, the present invention includes the following inventions.
(1) A method for evaluating a cell culture substrate having a substrate and a functional layer for culturing cells provided on the substrate,
Supplying droplets to the surface of the functional layer;
Injecting more liquid into the supplied droplet to expand the droplet or drawing liquid from the supplied droplet to shrink the droplet;
Measuring the contact angle of the expanding or contracting droplet;
Have
The cell culture substrate evaluation method, wherein a phenomenon in which the contact angle repeatedly increases and decreases is observed, and the state of the functional layer is evaluated based on the observation result.

(2) The method for evaluating a cell culture substrate according to (1), wherein the state of the functional layer is evaluated based on the number of times the contact angle repeats increasing and decreasing.
(3) The method for evaluating a cell culture substrate according to the above (1) or (2), wherein the functional layer contains a temperature-responsive polymer whose cell adhesiveness changes due to temperature change.
(4) An apparatus for carrying out the evaluation method according to any one of (1) to (3) above,
A stage on which the cell culture substrate is placed;
Droplet adjusting means for supplying droplets to the surface of the functional layer of the cell culture substrate disposed on the stage, and injecting liquid into the supplied droplets or sucking liquid from the supplied droplets; ,
An imaging means for imaging a droplet that expands by liquid injection or contracts by liquid suction;
A contact angle calculation unit that calculates a contact angle of a droplet based on an image photographed by an imaging unit;
An apparatus for evaluating a cell culture substrate.

(5) preparing a cell culture substrate having a substrate and a functional layer for culturing cells provided on the substrate;
For the prepared cell culture substrate, a step of performing the evaluation method according to any one of (1) to (3) above,
Have
The manufacturing method of the cell culture substratum which uses what the state of the functional layer was judged to be good as a result of evaluation.

  According to the present invention, it is not necessary to perform cell culture, and it is possible to evaluate the de-adhesiveness of cells on the cell culture substrate in a short time. In addition, it is possible to efficiently produce a cell culture substrate excellent in cell de-adhesiveness.

It is a figure for demonstrating the evaluation method of the cell culture substratum which concerns on this invention. It is a figure which shows one Embodiment of the evaluation apparatus of the cell culture substratum which concerns on this invention. It is a graph which shows the measurement result (room temperature) of the contact angle in Examples 1-3 and Comparative Example 1. It is a graph which shows the measurement result (room temperature) of the contact radius in Examples 1-3 and Comparative Example 1. It is a graph which shows the measurement result (40 degreeC) of the contact angle in Examples 1-3 and the comparative example 1. FIG. It is a graph which shows the measurement result (40 degreeC) of the contact radius in Examples 1-3 and the comparative example 1. FIG. It is the graph which compared the stick slip frequency measured in Examples 1-3 and the comparative example 1. FIG.

  Hereinafter, the present invention will be described in detail with reference to the drawings. First, the structure of the cell culture substrate which is the evaluation target of the present invention will be described.

  The cell culture substrate is configured by providing a functional layer for culturing cells on a substrate (usually, one side of the substrate). In general, the substrate and the functional layer are in direct contact with each other, but an intermediate layer such as a primer layer for facilitating the fixation of the functional layer may be interposed as necessary.

  The cell culture substrate is used by being installed on the inner bottom surface of a cell culture container that contains cells and a medium. Therefore, the base material may also serve as the bottom plate of the cell culture container, or the laminate of the base material and the functional layer is later joined to the inner bottom surface of the container formed from the bottom plate and the side wall. May be. When it is in the form of a laminate to be subsequently joined to the container, the substrate is plate-like or film-like, and the side opposite to the side on which the functional layer is provided is joined to the bottom plate of the cell culture vessel A pressure-sensitive adhesive layer and a release sheet can be appropriately provided.

  The material of the substrate is not particularly limited. Specifically, polyethylene terephthalate (PET), polystyrene (PS), polycarbonate (PC), TAC (triacetyl cellulose), polyimide (PI), nylon (Ny), low density polyethylene (LDPE), medium density polyethylene (MDPE) ), Resin materials such as polyvinyl chloride, polyvinylidene chloride, polyphenylene sulfide, polyether sulfone, polyethylene naphthalate, polypropylene, acrylic resin, and inorganic materials such as glass and quartz, and resin materials are preferably used.

  The surface of the substrate on the side where the functional layer is provided can be a surface subjected to an easy adhesion treatment. “Easy adhesion treatment” refers to treatment with an easy adhesive such as polyester, acrylic ester, polyurethane, polyethyleneimine, silane coupling agent, perfluorooctane sulfonic acid (PFOS), and the like.

  The material constituting the functional layer is not particularly limited as long as it has a function of culturing cells. For example, basic materials such as polylysine, basic polymers such as polylysine, aminopropyltriethoxysilane, N- (2-aminoethyl) -3-aminopropyltrimethoxysilane, etc., as materials showing adhesion to cells by electrostatic interaction Examples thereof include compounds and condensates containing them. In addition, as materials exhibiting adhesiveness to cells due to biological characteristics, fibronectin, laminin, tenascin, vitronectin, RGD (arginine-glycine-aspartic acid) sequence-containing peptide, YIGSR (tyrosine-isoleucine-glycine-serine-arginine) Examples include sequence-containing peptides, collagen, atelocollagen, gelatin and the like.

  Among them, when a cell culture substrate is used to detach cells from the substrate after cell culture or to collect cells in a sheet form, the cell adhesion by a predetermined stimulus is used as a functional layer. Those having a function of changing can be adopted. For example, a stimulus-responsive polymer that changes from cell adhesiveness to non-cell adhesiveness upon application of a stimulus can be included. Examples of the stimulus responsive polymer include a temperature responsive polymer, a pH responsive polymer, an ion responsive polymer, a photoresponsive polymer, and the like, and a polymer suitable for the cell sheet to be produced can be appropriately selected. . In particular, a temperature-responsive polymer is preferable because cell adhesion changes due to temperature change and stimulation can be easily applied.

  As the temperature-responsive polymer, for example, a polymer that exhibits cell adhesion at a temperature at which cells are cultured and exhibits cell non-adhesion at a temperature at which the produced cell sheet is peeled may be used. Specifically, at a temperature lower than the critical dissolution temperature, the affinity for surrounding water is improved, and the polymer takes in water and swells to show the property of making it difficult for cells to adhere to the surface (cell non-adhesiveness). It is preferable to use a material that exhibits a property (cell adhesion) that makes it easier for cells to adhere to the surface due to water detachment from the polymer at temperatures higher than the temperature. Such a critical solution temperature is called a lower critical solution temperature. A temperature-responsive polymer having a lower critical solution temperature T of 0 ° C. to 80 ° C., more preferably 0 ° C. to 50 ° C. may be used. This is because the cells can be stably cultured when T is 0 ° C to 80 ° C.

  Suitable temperature-responsive polymers include acrylic polymers or methacrylic polymers. More specifically, for example, poly-N-isopropylacrylamide (T = 32 ° C.), poly-Nn-propyl acrylamide (T = 21 ° C.), poly-Nn-propyl methacrylamide (T = 32 ° C.) Poly-N-ethoxyethyl acrylamide (T = about 35 ° C.), poly-N-tetrahydrofurfuryl acrylamide (T = about 28 ° C.), poly-N-tetrahydrofurfuryl methacrylamide (T = about 35 ° C.), and Examples include poly-N, N-diethylacrylamide (T = 32 ° C.).

  As a monomer for forming these polymers, a monomer that can be polymerized by irradiation with radiation can be used. Examples of the monomer include (meth) acrylamide compounds, N- (or N, N-di) alkyl-substituted (meth) acrylamide derivatives, (meth) acrylamide derivatives having a cyclic group, and vinyl ether derivatives. More than seeds can be used. When a single monomer is used alone, the polymer formed on the substrate is a homopolymer, and when multiple monomers are used together, the polymer formed on the substrate is a heteropolymer. Any form can be applied in the present invention.

  In addition, when it is necessary to adjust T depending on the type of proliferating cells, or when it is necessary to adjust the hydrophilic / hydrophobic balance of the functional layer, other monomers other than those described above may be further added and used in combination. Polymerization may be performed. Furthermore, you may comprise a functional layer using the graft | grafting or block copolymer of the said polymer and another polymer, or the mixture of the said polymer and another polymer. Moreover, it is also possible to crosslink within a range where the original properties of the polymer are not impaired.

  A functional layer containing a temperature-responsive polymer or the like can be formed on a substrate by appropriately adopting a conventionally known technique. For example, a coating composition containing a monomer that forms a target stimulus-responsive polymer by polymerization and an organic solvent capable of dissolving the monomer is prepared, and this is applied to the surface of a substrate according to a conventional coating method. A coating film is formed, and then the monomer in the coating film is polymerized by an appropriate means such as radiation irradiation to form a polymer, and a grafting reaction is performed between the surface of the substrate and the polymer. It can be formed by generating.

  The film thickness of the functional layer is, for example, preferably in the range of 0.5 nm to 300 nm, and particularly preferably in the range of 1 nm to 100 nm, but is not limited thereto.

  Next, the cell culture substrate evaluation method and evaluation apparatus of the present invention will be described. The evaluation method of the present invention includes a step of supplying droplets to the surface of the functional layer, and injecting liquid into the supplied droplets to expand the droplets, or sucking liquid from the supplied droplets to The method includes a step of shrinking the droplet and a step of measuring a contact angle of the droplet expanding or contracting.

Specifically, first, as shown in FIG. 1, an acicular tube S such as a microsyringe is preferably provided on the surface of the functional layer 20 in the cell culture substrate 1 including the substrate 10 and the functional layer 20. In use, the droplet L ₁ is supplied from the tip of the needle tube S. Subsequently, further injection of the liquid leaving the liquid droplets L ₁ in a state where the tip of the needle tube S into the droplet L ₁ is inserted, to expand the droplet. At this time, the droplet does not gently expand in proportion to the amount of liquid injected from the needle-shaped tube S while maintaining a constant contact angle, but does not interact with the droplet and the functional layer 20. This causes a phenomenon (stick-slip phenomenon) that discontinuously expands from the droplet L ₁ to the droplet L ₂ and the droplet L ₃ while repeating the contact angle once increasing and then decreasing. The inventor has found that the state of the functional layer 20 can be evaluated based on the observation result of the stick-slip phenomenon because the characteristic of the stick-slip phenomenon reflects the state of the functional layer 20. The stick-slip phenomenon is observed not only when the droplet expands but also when it contracts. Therefore, by supplying the droplet L ₃ in the beginning to the surface of the functional layer 20, then, the droplet L ₂ the liquid constituting the droplet by the needle-shaped tube member S by _suction, to a state of the droplet L ₁ The contact angle may be measured by gradually contracting and repeatedly decreasing and increasing in the contraction process. Water is usually used as the droplet L, but other liquids such as alcohol may be used in some cases. Specifically, ethanol, isopropyl alcohol, methanol or the like can be used.

  For example, when the functional layer 20 includes a temperature-responsive polymer such as poly-N-isopropylacrylamide (PIPAAm) and its immobilization amount is small, the hydrophobicity of the surface of the functional layer 20 is relatively high. Become. In that case, since the influence of the interaction between the droplet L and the functional layer 20 is small, when the droplet is expanded, the droplet is not easily held on the surface of the functional layer 20, and the contact angle once increased. Immediately decreases, and as a result, the number of times the contact angle is repeatedly increased and decreased within a predetermined time (referred to herein as the number of stick-slip times) increases. On the other hand, when the functional layer 20 contains a temperature-responsive polymer such as PIPAAm and the immobilization amount is large, the hydrophobicity of the surface of the functional layer 20 is relatively low (hydrophilicity is high). . In that case, since the influence of the interaction between the droplet and the functional layer 20 becomes large, when the droplet is expanded, the droplet is strongly held on the surface of the functional layer 20, and the contact angle of the droplet Does not start to decrease until a larger angle is reached, and as a result, the number of stick-slips within a certain time is reduced. Therefore, the state of the functional layer 20 such as the amount of immobilized polymer can be evaluated by measuring the number of stick slips within a certain time.

  Note that when measuring the number of stick-slip times, how much contact angle increase and decrease should be counted as one repeat unit can be unified among multiple cell culture substrates to be evaluated. Etc. can be set as appropriate. For example, a method in which only a change of 5% to 65% or more with respect to the current contact angle value within a unit time is recognized as an increase or decrease of the contact angle, or a change from increase to decrease within a predetermined time. Even if (or vice versa), there is a method of ignoring the variation and comparing the values at the first and last time points in the predetermined time range to recognize the tendency of increase or decrease. Alternatively, conventionally known peak detection methods in various technical fields may be used. In the embodiments described later, a stick-slip is defined as a period from when the contact angle increases to a decrease and then to an increase.

  In addition to the method of evaluating the state of the functional layer 20 based on the number of stick-slips, a method of observing a phenomenon in which the contact angle repeatedly increases and decreases (stick-slip phenomenon) and evaluating based on the observation result is possible. It can be adopted as appropriate. For example, an evaluation method focusing on the contact angle fluctuation cycle (average cycle) that repeatedly increases and decreases, and the average value of the contact angle difference (fall) until the contact angle starts from increasing to decreasing and then increases again The method etc. which evaluate based on etc. can be mentioned.

  The amount of droplets supplied to the surface of the functional layer 20 is not particularly limited as long as the contact angle of the droplets can be appropriately measured. Specifically, it is preferable that the minimum value and the maximum value of the droplet that expands or contracts with the measurement are in the range of 1 μL to 1 mL, particularly 1 μL to 20 μL. In addition, the injection / suction speed when liquid is further injected into the droplet or when the liquid is sucked can be appropriately set in consideration of measurement accuracy and efficiency. Specifically, it is preferably about 0.25 nL / msec to 0.5 μL / msec, particularly preferably about 0.5 nL / msec to 0.01 μL / msec.

  By using the above evaluation method in the cell culture substrate production process, it is possible to efficiently produce a cell culture substrate in which the state of the functional layer (immobilization amount, cell de-adhesiveness) is good. . Specifically, for example, cell culture is performed using some cell culture substrates in advance, and a suitable range (or threshold value) such as the number of stick-slip times by examining the correlation between the cell culture result and the number of stick-slip times, etc. To determine the quality of the product based on whether or not the values such as the number of stick slips of other cell culture substrates to be mass-produced thereafter are included in the above preferred range (or threshold). Can do. In this case, in the mass production process of the cell culture substrate, since conventional cell culture is not performed for the evaluation, the evaluation can be performed in an extremely short time, and the mass production process of the cell culture substrate is greatly efficient. Can be achieved. As an example, the substrate is made of polystyrene, the functional layer contains poly-N-isopropylacrylamide (PIPAAm), which is a temperature-responsive polymer, and 1 μL of water droplets are supplied to the surface of the functional layer at room temperature. When injecting water at an amount of 3 μL / msec to expand the water droplets, if the number of stick-slip times after the start of measurement is within the range of 2 to 4 times, the immobilized amount of PIPAAm and cell de-adhesion From the viewpoint of safety, it can be determined that the state of the functional layer is good.

  FIG. 2 shows an embodiment of an apparatus for performing the above-described evaluation method. This evaluation apparatus supplies a droplet L to the stage 11 on which the cell culture substrate 1 is disposed, and the surface of the functional layer of the cell culture substrate 1 disposed on the stage 11. A droplet adjusting means 12 for sucking a liquid from a droplet injected or supplied with a liquid; and an imaging means 14 for photographing a droplet L that expands or contracts by sucking the liquid. And a contact angle calculation unit 15a that calculates the contact angle of the droplet L based on the image photographed by the imaging means 14. In addition, for example, when a change in the contact radius of the droplet L that expands or contracts is also measured, a contact radius calculation unit 15b that calculates the contact radius of the droplet L based on the captured image can be further provided. Moreover, you may provide the temperature control means 13, such as a heater and a cooling device, as needed.

  The cell culture substrate 1 on the stage 11 is illuminated with light from the light source 16 that has passed through the diffusion plate 17. The imaging means 14 only needs to be able to capture the expanding or contracting droplet L, and specifically, a high-speed camera equipped with a CCD or the like is preferably used. As shown in FIG. 2, the droplet L can be photographed from the side of the stage 11 and the contact angle and contact radius of the droplet L can be measured. In addition, an imaging means is installed above the stage 11. The measurement accuracy may be improved by observing the droplet L in a plane and measuring the contact radius of the droplet L at the same time. The droplet adjusting means 12 includes a needle-like tube body such as a microsyringe that can be inserted into the droplet L, and a pump for injecting or sucking a set amount of liquid, and the needle-like tube body as a cell. A position adjustment mechanism for holding the culture substrate 1 at an appropriate position is appropriately provided. Furthermore, the contact angle calculation unit 15a and the contact radius calculation unit 15b have a function of analyzing the image acquired by the imaging unit and calculating the contact angle and the contact radius of the droplet L.

Next, the present invention will be described in more detail based on examples and comparative examples, but the present invention is not limited thereto.
(Example 1)
-Manufacture of cell culture substrate Poly-N-isopropylacrylamide (Aldrich) 2%, N-isopropylacrylamide was dissolved in isopropyl alcohol (IPA) to a final monomer concentration of 40% by weight to obtain a solution. Prepared. Subsequently, a polystyrene base material was used, the N-isopropylacrylamide solution was coated on the base material, electron beam irradiation was performed using an electron beam irradiation apparatus, and poly-N-isopropylacrylamide was applied to the base material. A functional layer made of (PIPAAm) was formed by graft polymerization. Thereafter, this laminate was washed with ion exchanged water at 5 ° C. and dried to obtain a cell culture substrate. The obtained cell culture substrate was sterilized with ethylene gas oxide.

-Evaluation of cell culture substrate The produced cell culture substrate was placed horizontally on the stage of a contact angle measurement device DM-501 (trade name, manufactured by Kyowa Interface Science Co., Ltd.), and the lower criticality of poly-N-isopropylacrylamide was measured. At room temperature (25 ° C.) that is lower than the melting temperature (T = 32 ° C.), 1.5 μL of pure water was dropped from the tip of the microsyringe onto the surface of the functional layer to form droplets. Subsequently, the liquid was further injected with the tip of the microsyringe inserted into the liquid droplet to expand the liquid droplet. The state of the expanding droplet was observed from the side with a CCD camera, and the contact angle θ and the contact radius r of the droplet were measured over time by software FAMAS (trade name, manufactured by Kyowa Interface Science Co., Ltd.). The measurement results are shown in FIGS. Further, the temperature condition was set to 40 ° C., which is higher than the lower critical solution temperature of poly-N-isopropylacrylamide, and the contact angle θ and the contact radius r of the expanding droplet were measured over time in the same manner as described above. The measurement results are shown in FIGS.

(Example 2)
The contact angle θ and the contact radius r of the droplets at room temperature and 40 ° C. were the same as in Example 1 except that the functional layer was formed using a solution in which the final monomer concentration was 35% by weight. Was measured. The measurement results are shown in FIGS.

(Example 3)
The contact angle θ and the contact radius r of the droplets at room temperature and 40 ° C. were the same as in Example 1 except that the functional layer was formed using a solution in which the final monomer concentration was 30% by weight. Was measured. The measurement results are shown in FIGS.

(Comparative Example 1)
The same measurement as in Example 1 was performed using a polystyrene base material on which no polymer or the like was fixed, and the contact angle θ and the contact radius r of the droplet at room temperature and 40 ° C. were obtained. The measurement results are shown in FIGS.

  As shown in FIGS. 3 and 5, for the cell culture substrates of Examples 1 to 3, a stick-slip phenomenon in which the contact angle θ of the droplet repeatedly increased and decreased was observed. Further, it was confirmed that the contact radius r increased in a stepped manner as shown in FIGS. Furthermore, with reference to the results of FIGS. 3 and 5, the number of stick slips (the number of peaks in each line in the graph) in which the contact angle θ repeatedly increases and decreases was counted for Examples 1 to 3 and Comparative Example 1. The number of times of stick-slip is the number of times within a range until 4700 msec elapses after the start of measurement. The result is shown in FIG. In addition, FIG. 7 has shown the average value of the result of having performed the measurement of Examples 1-3 and the comparative example 1 several times, respectively.

  As shown in FIG. 7, since Examples 1 to 3 differ in the number of stick-slips, the cell culture substrates of Examples 1 to 3 having different amounts of PIPAAm immobilized can be clearly distinguished by observing the stick-slip phenomenon. I understood. Further, in Example 1 where the amount of PIPAAm immobilized was large, the number of stick-slips was reduced. This is presumably because the surface of the functional layer becomes hydrophilic when the amount of immobilization increases, and the interfacial tension (force for holding droplets) becomes relatively large. On the other hand, in Example 1 with a small amount of immobilization, the surface of the functional layer becomes more hydrophobic and the interfacial tension is relatively small, so it is considered that the number of stick-slip times was observed. In the case of Comparative Example 1 in which the polymer is not fixed and does not have a functional layer, the hydrophobicity of the surface is the highest, so that the interaction between the droplet and the functional layer is weak, and the stick-slip phenomenon Was not observed. The contact radius r in Comparative Example 1 increases gently as shown in FIGS.

  Further, as shown in FIG. 7, the tendency that the number of stick-slips was the smallest in Example 1 and the largest in Example 3 was not changed regardless of whether the measurement temperature was room temperature or 40 ° C. From this, it became clear that the number of stick-slips reflects the state of the functional layer (polymer immobilization amount) regardless of the measurement temperature. Further, in all of Examples 1 to 3, the number of stick slips when measured at 40 ° C. was larger than that at room temperature. This is because, when the measurement temperature is higher than the lower critical solution temperature (T = 32 ° C.) of PIPAAm, water is desorbed from PIPAAm, and PIPAAm contracts to show cell adhesion, and the surface becomes more hydrophobic. This is considered to be because the interfacial tension (force for holding the droplets) becomes relatively small.

Next, the petri dish in which the cell culture substrate prepared in Examples 1 to 3 was placed on the inner bottom surface was filled with 10% FBS-containing DMEM medium, and 3T3 Swiss Albino cells were added at 6.25 × 10 ³ cells / cm ² . Sowing. After culturing at 37 ° C. for 18 hours, the cells were transferred to a low-temperature incubator at 20 ° C., and after incubation for 30 minutes, the state of cells detached and suspended was observed with a phase contrast microscope. As a result, the cell culture substrate of Example 1 had good cell detachability (removability), whereas the cell culture substrate of Examples 2 and 3 did not partially detach cells, and the detachability. Was not good. From these results, it was found that a cell culture substrate having good cell debonding property can be efficiently produced using the number of stick slips of the expanding droplet as an index.

1 cell culture substrate 10 substrate 11 stage 12 droplet adjusting means 13 temperature control means 14 imaging means 15a contact angle calculating section 15b contact radius calculation unit 16 light source 17 diffusion plate 20 functional layer _L, L 1 ~L ₃ droplets S needle tube r contact radius θ contact angle

Claims (5)

A cell culture substrate evaluation method comprising a substrate and a functional layer for culturing cells provided on the substrate,
Supplying droplets to the surface of the functional layer;
Injecting more liquid into the supplied droplet to expand the droplet or drawing liquid from the supplied droplet to shrink the droplet;
Measuring the contact angle of the expanding or contracting droplet;
Have
The cell culture substrate evaluation method, wherein a stick-slip phenomenon in which the contact angle repeatedly increases and decreases is observed, and the state of the functional layer is evaluated based on the observation result of the stick-slip phenomenon .   The method for evaluating a cell culture substrate according to claim 1, wherein the state of the functional layer is evaluated based on the number of times the contact angle repeats increasing and decreasing.   The method for evaluating a cell culture substrate according to claim 1 or 2, wherein the functional layer contains a temperature-responsive polymer whose cell adhesiveness is changed by temperature change. An apparatus for carrying out the evaluation method according to claim 1,
A stage on which the cell culture substrate is placed;
Droplet adjusting means for supplying droplets to the surface of the functional layer of the cell culture substrate disposed on the stage, and injecting liquid into the supplied droplets or sucking liquid from the supplied droplets; ,
An imaging means for imaging a droplet that expands by liquid injection or contracts by liquid suction;
A contact angle calculation unit that calculates a contact angle of a droplet based on an image photographed by an imaging unit;
An apparatus for evaluating a cell culture substrate. Preparing a cell culture substrate having a substrate and a functional layer for culturing cells provided on the substrate;
For the prepared cell culture substrate, a step of performing the evaluation method according to any one of claims 1 to 3,
Have
The manufacturing method of the cell culture substratum which uses what the state of the functional layer was judged to be good as a result of evaluation.

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